Borda Medical Technology Co. Ltd

Centrifuge sedimentation coefficient of determination

Centrifuge sedimentation coefficient determination is to analyze the most important purposes. Usually only need to tens of milligrams or even tens of micrograms of sample, preparation of a ~ 2 ml solution, into analysis of pools to the analysis of centrifugal few hours, they get a series of samples of centrifugal sedimentation diagram. According to the settlement plan can be used for qualitative analysis of the sample contained in the component can also be measured sedimentation coefficient of each component and the estimated molecular size and purity of the samples for inspection and heterogeneity determination in order to determine the relative content of components. Medical experimental

1. Principle clinical trial
Determination of sedimentation coefficient is a constant principle of the centrifugal force field samples of particle settling velocity were measured. Because the sample particles is small, can not directly see them to the settlement movement, so when the centrifugal speed interface of the sample particles as the sample, the average particle settling velocity. Schlieren and absorption of commonly used optical system to record the settlement map interface. In the settlement figure the general performance of the sample interface as a symmetrical peak, the highest point of the peak position of representatives of interface. Usually sedimentation coefficient measurement accuracy of ± 2%, but if the surface world graphics performance for the asymmetric peak-type, or would like to sedimentation coefficient measurement accuracy of ± 1% or less of the situation, according to the highest point of the peak position as the interface is not fully a time should use the second method from the position of interface.

2. Sedimentation coefficient determination of an instance of pharmacutical clinical contract research projects
⑴ Sample: Bovine serum albumin
⑵ the sample solution and centrifuge: According to the concentration of 0.6% bovine serum albumin dissolved in the 0.14M NaCl, 0.01M phosphate buffer, pH7.0. Slot analysis using 12mm thick pool, while adding solution while adding solvent, about the 0.3ml. Analysis of pools pool balance of weight and balance, so that a balanced pool of 0.5g or less than the analysis of pools of light, and then into analysis of rotor, respectively. Analysis of centrifugal rotor mounted on an analysis of why turning off cavity vacuum. Open Schlieren light source, select the operating speed of a 60000 rev / min, centrifugation at room temperature. After rotating the vacuum chamber to start running away from a machine to accelerate at this time can be seen in the observation window centrifugal graphics. After reaching the operating speed for constant speed centrifuge. Yet to see samples of cutting-edge peaks after 6-minute intervals can be photographed, according to a total of six. According to end-phase can be shut down, remove the sample solution, cleaning brick and analysis of pools. Rinse with a strong contrast between the imaging camera after the settlement was Schlieren optical graphic photos.

⑶ settlement image measurement: Schlieren settlement figure can be compared with a long instrument, reading microscope, or projector measurement. Requirements to measure the horizontal range of 6cm, measurement accuracy should be better than 10μm. Usually reading microscope has been able to meet the requirements. Projector can make images larger on the screen, read more easily measured, eye fatigue. Measured at the time of settlement of the image on film placed on the measuring instruments, so that the vertical line and the liquid level measuring instrument in the vertical lines overlap, and then followed by measuring restricted reference materials cross marking hole, liquid level, interface Feng Jian, and the outer reference holes the location of each image measured at least read three times, and averaged. In turn to each image in the same way according to measurements, the data list made 5. Rowpar Pharmaceuticals, Inc.

Photo ID x1 (mm) x2 (mm) x3 (mm) x4 (mm) (x3-x1) / a log [5.65 + (x3-x1) / a]
1 45.426 37.626 33.807 13.810 0.6125 0.7967
2 45.640 37.830 33.351 13.420 0.6485 0.7992
3 45.890 38.070 32.895 13.663 0.6854 0.8017
4 45.733 37.921 32.163 13.523 0.7161 0.8039
5 45.802 38.000 31.580 13.593 0.7507 0.8062
6 46.009 38.211 31.172 13.800 0.7830 0.8084

(4) The calculation of sedimentation coefficient pharmaceutical outsourcing

S: measuring the settlement plan, first check the map and one sixth of each graph the value of x2-x1, 2 graphs the difference between the value of x2-x1 interface should be less than the peak position of the measurement error . X2-x1 measured the difference of 0.002, indicating liquid level in the centrifuge within 30 minutes change of 0.002mm. Interface peak within 30 minutes distance is the sixth mobile number graph x1-x3 by No. diagram x1-x3, is 3.208mm, the peak level measurement means allowing 0.03. Inland surface changes can be 0.002mm peak measurement error is much smaller than 0.03mm, shows no leakage during centrifugation.
By a = (x4-x1) / 1.7 Calculating the optical path of the analysis of the pool magnification, and then press (x3-x1) / a peak demand to leave the operator interface, the actual distance within the reference hole, and then x = 5.65 + (x3 – x1) / a operator interface for peak demand to leave the center of rotation of the actual distance, then the number of tables on the demand by the plant operator logx, calculated data are also listed in Table 5.

3. Centrifugal Image pharmaceutical Analysis
When the centrifugal beginning to see a quick settlement if the peaks within a few minutes to reach the bottom of the pool analysis, most people took place due to sample some of polymerization of the polymer to form rapid subsidence, such as the protein sample solution to certain types of variability factors I know some degeneration of the precipitation. Centrifugation after the samples up to speed in mind that heart images show a symmetrical peak shape, can generally be considered homogeneous sample is centrifuged. However, the true homogeneity of samples was also used other ways to further testing, such as electrophoresis, chromatography and so on. Some mixed samples will also be given a chance symmetrical peak. Peaks will normally extend over time, which is due to the spread of the results of the sample. However, if the peak shape extended quickly, then the sample may be more scattered in nature. If the images in the performance of centrifugal several peaks, indicating the sample has several components, each peak represents the corresponding component of the settlement interface, so you can determine each component of the settlement coefficient. According to peak area of component concentration can be measured.
Sometimes the images show a centrifugal asymmetric peak, which may be caused due to the following types of situations. ① several sedimentation coefficient close to the component peaks overlap, ② sample is polydisperse in nature, and its molecular weight distribution uneven, ③ some strong interaction between polymer, its velocity is greatly dependent on the concentration, such as DNA, polysaccharides elements, if measured in high concentration, the concentration in the interfacial region caused by changes in velocity due to inconsistencies in peak shape was asymmetrical distribution of Er Zhi. A greater concentration of these samples even the formation of a very sharp interface, in the performance of centrifugal sedimentation diagram into a vertical line, do not see any peak shape. According to this graphic determination of sedimentation coefficient error will be high. Usually such samples should be measured at very dilute concentrations of these conditions, smaller molecular interaction, interface extended peak shape, it can be measured to the correct sedimentation coefficient.

4. Determination of sample purity and concentration of pharmaceuical R&D
Centrifuges can be measured analysis of the purity and concentration of the sample, it is also an analysis of centrifugal purposes. Samples are usually centrifugal graphics performance when a symmetrical peak shape may be that the sample is centrifuged homogeneous. However, when we must pay attention to the purity determination of the determination of the concentration of the sample should be determination of the concentration of the sample is greater than the sensitivity of determination for more than a hundred times to measure meaningful. For example, Schlieren optical determination of the sensitivity of the sample 0.1mg/ml, then the purity of the sample when measuring the concentration should be 90mg/ml. If the selected sample 1mg/ml concentration determination, the settlement even if the graphics performance of a single peak, still unable to determine its purity is 0% or 99%. If the sample is multi-component, the performance of centrifugal diagram of several peaks of each peak area and the total area of all peaks can be the ratio of the relative concentration of each component. Should be considered in calculating the dilution effect of radiation, that is the size of the value of the various components in their positions according to the formula diluted with radiation correction to the surface at a value, and then calculate the relative. Also note that the concentration of this estimate is based on the refractive index of the various components have the same ratio based on the assumption that. Area under the peaks with the concentration and refractive index of the component ratio and instruments can also calculate the group Constant sweep the absolute concentration. Roche in the UK

5. Band sedimentation method research Organization
Ribbon similar to the rate of sedimentation zones away from the miscellaneous and. Approach the use of synthetic interface pool, in the pool pre-accession 0.55ml of 1M NaCl solution, and then to 2000 r / min rotational speed lower berth on a 0.01ml sample solution, and then accelerated to 30,000 rev / min, with absorption optical records of the sample settlement graphics. As NaNl to the sample, the proliferation of products with the formation of a density gradient zone, the sample can be stabilized settlement zone. If there are multiple samples sedimentation coefficient of different components, they will be presented in accordance with their respective velocity sedimentation zone. Use of this method can test the sample purity, estimate the relative content of the composition and sedimentation coefficient. Applicable to small amounts of nucleic acid and virus samples analyzed by HPLC.Roche

Centrifuge is the use of centrifugal force on the mixture (containing solids) for separation and precipitation of a special instrument. Laboratory centrifuges are commonly used electric low-speed, high-speed centrifuge and low-speed, high-speed refrigerated centrifuge, as well as speed analysis, prepared by a variety of models of dual-use refrigerated centrifuge. Among them, low-speed (including large capacity) and high-speed refrigerated centrifuge centrifuges most widely used is the preparation of biological laboratory used for the separation of biological macromolecules essential and vital tool. In the experiment, purports to separate the sediment and liquor, often the use of filtering and centrifugation two methods. However, in the following circumstances, the use of centrifugal method effective. (Pharmacutical clinical contract research projects trade platform – www.crolegacy.com)
① precipitation sticky or viscous mother liquor.
② precipitation particles is small and easy through the filter paper.
③ excessive amount of loose sediment.
④ small amount of precipitation needed quantitatively. Or liquor a very small number, separation should be to reduce losses.
⑤ precipitate and mother liquor must be rapidly separated.
⑥ general colloidal solution.